Many bacterial cytolytic toxins exhibit an amphimorphic nature in which they are produced as soluble monomers, but ultimately end up as membrane-associated oligomers. These toxins lack obvious transmembrane domains and in fact have relatively hydrophilic structures. The mechanism by which these proteins carry out the transition from a soluble protein to a membrane associated complex has not been identified. Unlike many bacterial toxins, such as diphtheria toxin or the colicins, pH does not act as a trigger for the formation of a "molten globule" or insertion intermediate. Perfringolysin O (PFO), a cytolysin (Mr 54,000) produced and secreted by Clostridium perfringens, belongs to a family of related cytolysins collectively called the "thiol-activated cytolysins" that are produced by a variety of gram positive pathogenic bacterial species. PFO typifies a cytolytic toxin which has a hydrophilic primary structure but forms a cytolytic membrane complex. After binding to the target membrane, PFO monomers oligomerize into supramolecular complexes and lyse the cell. How membrane insertion and pore formation by PFO is accomplished remains unknown. This proposal is designed to identify the location (i.e., protein-aqueous, protein-membrane, or protein-protein) of various domains of PFO before and after its interaction with target membranes. The specific aims of this proposal are: 1) generation of single-site cysteine substitutions in PFOala, 2) identification of PFO-membrane interactions; 3) identification of regions of PFO exposed to the aqueous medium at various stages of the cytolytic mechanism; 4) identification of residues located at the interfacial domains of the monomeric subunit of the PFO oligomer; and 5) determination of the extent of PFO insertion into the membrane at various stages of the cytolytic process. Unique cysteines will be placed into the primary structure of PFO to act as specific attachment sites for fluorescent dyes such as NBD whose emission is sensitive to the polarity of their environment. The fluorescence lifetime intensity and collisional quenching of the single dye attached to PFO will be monitored to determine if a specific residue remains is the aqueous phase, moves into the lipid bilayer, or forms part of the interfacial domains that are in contact in the oligomerized PFO. Since oligomerization only occurs above 10C, whereas binding occurs at all temperatures, insertion of a probe labeled residue into the bilayer can be assigned to either the binding event or the oligomerization event.